Fixed biological samples are routinely impregnated and embedded in paraffin wax or paraffin blends to preserve them for later analysis. Nucleic acid isolation from these samples is usually performed after the paraffin wax or paraffin blend has been removed by extraction with organic solvents such as toluene, xylene, limonene, or other suitable solvent. These organic solvents are very volatile and require special processing such as use of ventilated hoods and special waste disposal. The use of these organic solvents increases the cost of analysis and exposure risk associated with each sample tested and has serious negative effects for the environment. Further, use of such organic solvents is not amendable to high throughput isolation and analytical methods.
Alternate methods provide for heating the paraffin embedded sample such that the paraffin melts and is physically separated from nucleic acid. See, for example, U.S. Published Patent Application No. 2008/0050746. U.S. Pat. No. 7,410,753 relates to heating a biological sample and a paraffin embedding media above the melting point of the paraffin embedding media, and rinsing with a paraffin embedding media-immiscible fluid in order to remove the heated paraffin embedding media from the biological sample, and replacing the paraffin embedding medium with a fixation solution.
Embodiments herein provide for in situ nucleic acid isolation from biological samples embedded in a hydrophobic matrix such as paraffin or a paraffin-blend. No separate step of removing embedding medium is used. Further, the embodiments presented herein are automation friendly and enable high throughput processing of said samples.